Ovis BPI Gene Cloning, Expression And Activity Detection

Bactericidal/permeability-increasing protein (bactericida/permeability increasing protein, BPI) ispresent in human and mammalian polymorphonuclear cells, a cationic antimicrobial protein, in manyantibacterial ingredients in neutrophils, BPI protein is the only non-specific anti-Gram-negative bacteriaand is currently found in endotoxin, but also has immunomodulatory effects of antimicrobial substances. Inthis study, Xinjiang Bashibai sheep and hybrid sheep for the study, using molecular biology techniques BPIfull-length cDNA clone genes and sequence analysis, expression and purification of BPI protein, and thentest to verify its biology through inhibition activity.Objective: This study Bashibai BPI cDNA cloning full-length sequence of sheep and goat hybrid gene,its bioinformatics analysis through prokaryotic expression to Bashibai sheep and hybrid sheep BPIgenesheep and goat BPI hybrid gene, recombinant BPI protein inhibitory effect of Gram-negative bacteria,verify its biological activity, to lay the foundation for further study the biological function of sheep BPIprotein.Methods:(1) amplification and sequence Bashibai sheep and goat BPI length cDNA hybridizationanalysis. According to BPI NCBI gene sequence of bovine reported, design degenerate primers, clonedsheep and hybrid sheep Bashibai BPI gene fragments, fragments through the design RACE primers wereamplified by RACE cDNA sequence above two sheep BPI gene and sequencing, splicing, and finally theuse of biological software resulting BPI cDNA sequence analysis.(2) biological information Bashibaisheep and goat hybrid BPI Gene cDNA analysis: the use of online analysis tools BPI gene sequencesobtained were analyzed gene structure and physical and chemical characteristics, and predict its function;using a network database and procedures BPI protein for bioinformatics analysis.(3) Bashibai sheep andgoat BPI hybrid gene amplification of the N-terminal sequence: hybridization capture Bashibai sheep andgoat peripheral blood, neutrophils isolated and extracted RNA, RACE technology is based on the BPIcDNA sequences of specific of PCR primers were cloned sheep Bashibai hybrid sheep BPI N-terminalsequence of its gene, the PCR product recovery pMD18-T vector and then transformed into competentcells DH5α, testing positive clones were sequenced.(4) Construction and expression Bashibai sheep andgoat BPI hybrid gene expression vectors: the correct Bashibai hybrid sheep and goat identification BPI N-terminal sequence is connected to the prokaryotic expression vector pGEX construct pGEX-BPI expressionplasmid transformation to competent cells BL21(DE3), the optimized conditions induced expression andSDS-PAGE analysis and identification.(5) Bashibai sheep and goat hybrid BPI protein purification andbiological activity detection: the use of GST resin purification methods respectively Bashibai sheep andgoat hybrid recombinant BPI protein purification, SDS-PAGE detection. Respectively broth dilutionmethod and agarose antibacterial test detection of two sheep were recombinant BPI protein biologicalactivity.Results and conclusions:(1) Bashibai sheep BPI full-length cDNA of1922bp, which open readingframe1452bp, encoding483amino acids of both sides of the coding region of each wing has a5′-UTR54bp and the3′-UTR416bp. BLAST results table thanks to Yang Ming Bashi BPI and sheep (forecast),cows, killer whales, wild boar, monkeys, human, rabbit, mouse, Xenopus nucleotide homology were99%,91%,78%,74%,64%,61%,58%,53%,50%. BPI hybrid sheep full length cDNA of1939bp,which open reading frame1452bp, encoding483amino acids of both sides of the coding region of eachwing has a5′-UTR84bp and3′-UTR403bp. BLAST results show that BPI hybrid sheep and sheep(forecast), cows, killer whales, wild boar, monkeys, human, rabbit, mouse, Xenopus nucleotidehomology were97%,91%,77%,74%,64%,60%,58%,52%,50%. Compare Bashibai BPI hybridsheep after sheep and open reading frame found that11base changes, including changing the sixnucleotides encoding amino acids making it a difference.(2) Bashibai sheep BPI gene ORF in (A+T) content of45.25%[657],(C+G) content of4.75%[795]; their hybrid sheep BPI gene ORF in (A+T)content was44.97%[653],(C+G) content of55.03%[799]. Two kinds of sheep BPI protein arehydrophilic proteins, secreted proteins are mainly present in the extracellular matrix; Bashibai sheep BPIprotein contains19α helices,18β fold,33corners and random coil, and BPI hybrid protein containing18goat α helix. N-terminal signal peptide were there BPI1and BPI2two conserved domains. BPI genephylogenetic study found Bashibai sheep/hybrid sheep in sheep, cows, killer whales, wild boar, human,monkeys, rabbits, mice, Xenopus laevis, and the sheep first clustered together by molecules, after andcattle clustered into one class, which is consistent with animal taxonomy.\3) Bashibai sheep and goathybrid BPI N-terminal sequence of the gene are598bp, encoding198amino acids. By comparing the twoN-terminal sequence of sheep and found a total of three nucleotide differences between the two, the firsthybrid sheep coding sequence328, in the first codon of G into T, resulting in an amino acid Gly (glycine)into a Cys (cysteine); section449, in the second position of codon into T G, resulted in the amino acid Val(valine) into Gly (glycine); section660, in codon a to second position G, resulted in the amino acid Gln(glutamine) into Arg (arginine).(4) successfully constructed Bashibai BPI hybrid sheep sheep andprokaryotic expression vector and expressed in E. coli host strain expressing a fusion protein molecularweight of about46kD.(5) the expression product was purified by SDS-PAGE analysis showed thatobserved with the single purpose of bands Bashibai successfully purified recombinant BPI sheep and goathybrid protein. Broth dilution method with the results show that the protein concentration increases, theeffect of enhancing the role of E. coli, and maintain optimal activity at4h; agarose antibacterial testsshowed Bashibai sheep BPI protein than the hybrid sheep BPI protein antibacterial effect stronger, and ithas antibacterial effect on E. coli, Staphylococcus aureus without inhibition. Description of the experimentexpressed Bashibai sheep and goat hybrid recombinant BPI protein has good biological activity.